RESUMO
Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/ultraestrutura , Proteínas de Ligação a DNA/genética , Sequências Hélice-Alça-Hélice/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/ultraestrutura , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Sítios de Ligação/genética , Proteínas de Ligação a DNA/química , Proteínas de Homeodomínio/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Fosforilação/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Fatores de Transcrição HES-1RESUMO
Ectodomain shedding of membrane receptors and ligands carried out by ADAMs (A disintegrin and metalloprotease) plays a major role in several signaling pathways, including Notch. The grounds of substrate recognition, however, are poorly understood. We demonstrate that a recombinant protein corresponding to the juxtamembrane region of Jagged-1, one of the Notch ligands, behaves as a structured module and is cleaved by ADAM17 catalytic domain at E1054. A short synthetic peptide is cleaved at the same site but at a much higher rate, implying that the structure of the cleavage site in the native protein is a key determinant for substrate recognition. We also show that an Alagille syndrome-associated mutation near E1054 increases the cleavage rate, which suggests that this mutation may lead to an unbalance in Notch signaling due to a higher level of Jagged-1 shedding.
Assuntos
Proteínas ADAM/química , Proteínas de Ligação ao Cálcio/química , Domínio Catalítico , Peptídeos e Proteínas de Sinalização Intercelular/química , Proteínas de Membrana/química , Proteólise , Proteína ADAM17 , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Ligantes , Proteínas de Membrana/genética , Dados de Sequência Molecular , Receptores Notch/metabolismo , Proteínas Serrate-JaggedRESUMO
HES-1 is a transcriptional repressor of the basic helix-loop-helix (bHLH) family and one of the main downstream effectors in Notch signaling. Its domain architecture is composed of a bHLH region, an Orange domain, and a poorly characterized C-terminal half. We show that different degrees of structural order are present in the different regions of HES-1. The isolated bHLH domain is only marginally stable in solution, and partially folds upon dimerization. Binding to DNA promotes folding, stabilization, and protection from proteolysis of the bHLH domain. The Orange domain, on the contrary, is well folded in all conditions, forms stable dimers, and greatly increases protein resistance to thermal denaturation. The isolated proline-rich C-terminal region is mainly disordered in solution, and remains unstructured also in the full length protein. Measurements of binding constants show that HES-1 recognizes dsDNA synthetic oligonucleotides corresponding to several functional DNA targets with high affinity, but with relatively little specificity. We propose that order/disorder transitions in the different domains are associated not only with binding to DNA, but also with protein homo- and hetero-dimerization.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Proteínas de Homeodomínio/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Dicroísmo Circular , DNA/química , DNA/genética , DNA/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Desnaturação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Temperatura , Fatores de Transcrição HES-1RESUMO
Notch signaling plays a key role in cell differentiation and is very well conserved from Drosophila to humans. Ligands of Notch receptors are type I, membrane spanning proteins composed of a large extracellular region and a 100-150 residue cytoplasmic tail. We report here, for the first time, the expression, purification, and characterization of the intracellular region of a Notch ligand. Starting from a set of synthetic oligonucleotides, we assembled a synthetic gene optimized for Escherichia coli codon usage and encoding the cytoplasmic region of human Jagged-1 (residues 1094-1218). The protein containing a N-terminal His(6)-tag was over-expressed in E. coli, and purified by affinity and reversed phase chromatography. After cleavage of the His(6)-tag by a dipeptidyl aminopeptidase, the protein was purified to homogeneity and characterized by spectroscopic techniques. Far-UV circular dichroism, fluorescence emission spectra, fluorescence anisotropy measurements, and (1)H nuclear magnetic resonance spectra, taken together, suggest that the cytoplasmic tail of human Jagged-1 behaves as an intrinsically unstructured domain in solution. This result was confirmed by the high susceptibility of the recombinant protein to proteolytic cleavage. The significance of this finding is discussed in relation to the recently proposed role of the intracellular region of Notch ligands in bi-directional signaling.